Intervertebral disc (IVD) degeneration is a primary or secondary cause of low back pain with associated medical costs ranging from at least $20 to $100 billion annually, yet surgical treatment options do not focus on preventing degeneration or repairing discs. The high prevalence of disc degeneration in humans and rarity of disc degeneration in animal species that retain notochordal (NC) cells into maturity supports the concept that NC cells are integral to preventing degeneration and/or promoting repair. There are few studies investigating interactions between NC cells with mature IVD cells, even fewer exploring the mechanisms for these interactions, and no scientific literature on interactions between NC cells with bone mesenchymal stem cells (MSCs). The first hypothesis is that NC cells orchestrate the biology of IVDs, functioning by interacting with and influencing the functions of nucleus pulposus (NP) and annulus fibrosus (AF) cells in a dose-dependent manner that is mediated via soluble factors. The second hypothesis is that NC cells promote differentiation of MSCs towards a young NP phenotype, via the action of soluble factors. The proposed Specific Aims will test the functional relevance and mechanisms of interaction between soluble factors from NC cells with NP and AF cells (Aim 1) and with MSCs (Aim 2). The assay system will isolate soluble factor effects, elucidate mechanisms for action in 3-dimensional pellet cultures that maintain cell morphology, and specifically evaluate the role of connective tissue growth factor (CTGF). The parameters to be analyzed include cell biosynthesis and phenotypic differentiation based on IVD matrix gene expression, protein synthesis, and cell proliferation. NC, NP and AF cells are harvested from immature porcine and mature bovine IVDs as these large animal models provide a consistent source of sufficient numbers of cells required in the proposed studies. Human MSCs will be used in view of their clinical relevance. Preliminary results demonstrate significant interactions of NC cells with mature IVD cells, evidence that NC cell soluble factors strongly increase glycosaminoglycan production in MSCs and differentiate these cells towards a novel chondrogenic phenotype with characteristics of young NP cells, and that CTGF is present in NC conditioned medium. The exploration of a young NP phenotype, evaluation of NC conditioned medium effects on IVD cells and MSCs, and the determination of specific growth factors responsible for these effects are consistent with the exploratory/high impact objectives of the R21 mechanism. Clinical relevance involves determination of target molecules for pharmaceutical use on mature disc cells and on MSCs to prevent degenerative changes and promote cell therapy repair techniques. PUBLIC HEALTH RELEVANCE: Intervertebral disc degeneration is commonly implicated in low back pain with a very high prevalence in humans and very low prevalence in animal species that retain notochordal cells into maturity. Basic science information on notochordal cells is very limited, and the overall hypothesis is that notochordal cells orchestrate the biology of the intervertebral disc and may be used to prevent disc degeneration and to promote disc repair. The purpose of the proposed studies is to investigate the functional relevance and mechanisms of interaction between notochordal cells with nucleus pulposus and anulus fibrosus cells and also with bone mesenchymal stem cells.